Composition containing a peptide and a pigment and the use thereof in darkening the skin

ABSTRACT

The present invention relates to compositions containing a peptide(s) and a pigment(s) and the use thereof in darkening the skin.

FIELD OF THE INVENTION

The present invention relates to compositions containing a peptide(s)and a pigment(s) and the use thereof in darkening the skin.

BACKGROUND OF THE INVENTION

The darkening of skin color is a concern for many individuals. Mostpeople obtain darker skin through exposure to UV light (e.g., suntanningor UV lamps). UV exposure, however, results in accelerated skin agingand increased incidence of skin cancer. The ability to generate a tannedappearance without incurring photodamage, thus, is important to manyindividuals. Accordingly, alternative methods for “sunless tanning” haveevolved.

One method is the use of products containing dihydroxy acetone (DHA).Some of these products, however, produce color that is too orange andunnatural to the user. Moreover, the DHA-produced skin color onlyminimally protects the user from UV irradiation. Products containingbeta-carotene and cantaxanthin have also been used to darken the skin.These products, however, also result in unnatural skin color and reducedsun-protection as compared to naturally tanned skin. Thus, a product isdesired that could enhance the body's natural pigment content, resultingin a desired skin color and enhanced photo-protection, without the needof UV exposure.

SUMMARY OF THE INVENTION

The present invention relates to compositions containing a peptide(s)and a pigment(s) and the use thereof in darkening the skin.

Other features and advantages of the present invention will be apparentfrom the detailed description of the invention and from the claims

DETAILED DESCRIPTION OF THE INVENTION

It is believed that one skilled in the art can, based upon thedescription herein, utilize the present invention to its fullest extent.The following specific embodiments are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference. Unless otherwise indicated, a percentagerefers to a percentage by weight (i.e., % (W/W)).

Definitions

What is meant by “darkening the skin” is darkening the appearance of theskin, including, but not limited to, tanning the skin.

As used herein, “topical applying” means directly laying on or spreadingon outer skin using, e.g., by use of the hands or an applicator such asa wipe, roller, or spray.

As used herein, “cosmetically-acceptable” means that the peptides,pigments, or inert ingredients which the term describes are suitable foruse in contact with tissues (e.g., the skin) without undue toxicity,incompatibility, instability, irritation, allergic response, and thelike.

As used herein, “safe and effective amount” means an amount of thepeptide or composition sufficient to induce a darkening of the skin, butlow enough to avoid serious side effects. The safe and effective amountof the compound or composition will vary with the area being treated,the age and skin type of the end user, the duration and nature of thetreatment, the specific compound or composition employed, the particularcosmetically-acceptable carrier utilized, and like factors.

Pigment

What is meant by a “pigment” is a compound(s) that can be taken up byepidermal cells in the presence of the peptides described below,resulting in visually darker look to the skin. Examples of such pigmentsinclude, but not limiting to, melanin and melanin derivatives (e.g, bothmelanin polymers and lower molecular weight water-soluble melaninderivatives); extracts from natural sources containing pigments (e.g.,brown pigments from plants from the Hedychium genus or Bearberry genusor yellow, orange and red pigments, from plants containing carotenoidsor canthaxanthins); or synthetic chemicals such as compounds containingcopper (e.g., copper salts such as CuCl₂) or synthetic carotenoids orcanthaxantins. What is meant by an “extract” is a mixture of compoundsisolated from a natural source (e.g., a plant). Examples of syntheticmelanin derivatives are disclosed in U.S. Pat. Nos. 5,618,519,5,384,116, and 5,227,459. Examples of soluble melanin derivatives aredisclosed in U.S. Pat. Nos. 5,744,125, 5,225,435, 5,218,079, and5,216,116. Examples of commercially available soluble melaninderivatives include Melasyn-100™ from San-mar laboratories, Inc.(Elmsford, N.Y.) and MelanZe™ from Zylepsis (Ashford, Kent, UnitedKingdom).

The amount of pigment present in the composition will depend on the typeof pigment used. The pigment typically will be present in thecomposition in an amount from about 0.001% to about 20% by weight, inparticular in an amount from about 0.005% to about 5% by weight.

Peptides

The composition of the present invention comprises a peptide of theformula I

-   -   R₁        >A₁-A₂-A₃-A₄-A₅-A₆-A₇-R₃  Formula I    -   R₂    -   wherein:        -   A₁ is Ser or 2,3-diaP, or is absent;        -   A₂ is Val, Leu, Ile, or Cha;        -   A₃ is Val, Leu, Ile, or Cha;        -   A₄ is Gly or Ala;        -   A₅ is Lys, Arg, or Har;        -   A₆ is Val, Leu, Ile, or Cha, or is absent;        -   A₇ is Asp or Glu, or is absent; provided, A₇ is absent if A₆            is absent;        -   each R₁ and R₂, independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀            phenylalkyl, or C(=O)E₁, where E₁ is C₁₋₂₀ alkyl, C₃₋₂₀            alkenyl, C₃₋₂₀ alkynyl, phenyl, 3,4-dihydroxyphenylalkyl,            naphthyl, or C₇₋₁₀ phenylalkyl; provided that when either R₁            or R₂ is C(=O)E₁, the other must be H; and        -   R₃ is OH, NH₂, C₁₋₁₂ alkoxy, C₇₋₁₀ phenylalkoxy, C₁₁₋₂₀            naphthylalkoxy, C₁₋₁₂ alkylamino, C₇₋₁₀ phenylalkylamino, or            C₁₁₋₂₀ naphthylalkylamino;    -   or a cosmetically acceptable salt thereof.

In one embodiment, R₁ and R₂, which are bound to the N-terminus of thepeptide, are both H. In another embodiment, R₁ is H and R₂ is C(=O)E₁(e.g., palmitoyl, oleatoyl, or stearatoyl).

Examples of peptides of the present invention include, but are notlimited to, H₂-Leu-Ile-Gly-Arg-NH₂ (Peptide 1; SEQ.ID.No.1),H₂-Leu-Ile-Gly-Arg-OH (Peptide 2; SEQ.ID.No.2),H₂-Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (Peptide 3; SEQ.ID.No.3), andH₂-Ser-Leu-Ile-Gly-Arg-Leu-OH (Peptide 4; SEQ.ID.No.4),Palmitoyl-Leu-Ile-Gly-Arg-NH₂ (Peptide 5; SEQ.ID.No.5),Palmitoyl-Leu-Ile-Gly-Arg-OH (Peptide 6; SEQ.ID.No.6),Palmitoyl-Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (Peptide 7; SEQ.ID.No.7),Palmitoyl-Ser-Leu-Ile-Gly-Arg-Leu-OH (Peptide 8; SEQ.ID.No.8),Stearatoyl-Leu-Ile-Gly-Arg-NH₂ (Peptide 9; SEQ.ID.No.9),Stearatoyl-Leu-Ile-Gly-Arg-OH (Peptide 10; SEQ.ID.No.10),Stearatoyl-Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (Peptide 11; SEQ.ID.No.11), andStearatoyl-Ser-Leu-Ile-Gly-Arg-Leu-OH (Peptide 12; SEQ.ID.No.12), or acosmetically-acceptable salt thereof.

The symbol A₁, A₂, or the like used herein (e.g., in FIG. 1) stands forthe residue of an alpha-amino acid. Such symbols represent the generalstructure, —NH—CH(X)—CO— or ═N—CH(X)—CO— when it is at the N-terminus or—NH—CH(X)—CO— when it is not at the N-terminus, where X denotes the sidechain (or identifying group) of the alpha-amino acid, e.g., X is—CH(CH₃)₂ for Val. Note that the N-terminus is at the left and theC-terminus at the right in accordance with the conventionalrepresentation of a polypeptide chain. R₁ and R₂ are both bound to thefree nitrogen atom N-terminal amino acid (e.g., A₁ or A₂) and the R₃ isbound to the free carboxy group of the C-terminal amino acid (e.g., A₅,A₆, or A₇).

“Cha” herein refers to cyclohexylalanine, “2,3-diaP” refers to2,3-diaminoproprionic acid, and “Har” refers to homoarginine.Furthermore, where the amino acid residue is optically active, it is theL-form configuration that is intended unless the D-form is expresslydesignated. An alkyl group, if not specified, contains 1–12 carbonatoms.

The peptide of the invention can be provided in the form of cosmeticallyacceptable salts. Examples of preferred salts are those withtherapeutically acceptable organic acids, e.g., acetic, palmitic, oleic,stearic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic,salicylic, methanesulfonic, or pamoic acid, as well as polymeric acidssuch as tannic acid or carboxymethyl cellulose, and salts with inorganicacids such as the hydrohalic acids (e.g., hydrochloric acid), sulfuricacid or phosphoric acid.

The amount of peptide present in the composition will depend on thepeptide used. The peptide typically will be present in the compositionin an amount from about 0.001% to about 10% by weight, in particular inan amount from about 0.005% to about 5% by weight.

The method for synthesizing peptides of the present invention are welldocumented and are within the ability of a person of ordinary skill inthe art. See, e.g., Bodanszky M, Int J Pept Protein Res 25(5):449–74(1985), Fmoc Solid Phase Peptide Synthesis, eds. Chan, W. & White, P.(Oxford University Press, 2000), and Chemial Approaches to the Synthesisof Peptides and Proteins, Lloyd-Williams, P. et al. (CRC Press, 1997).

Topical Compositions

The topical compositions useful in the present invention involveformulations suitable for topical application to skin. In oneembodiment, the composition comprises the peptide, pigment, and acosmetically-acceptable topical carrier. In one embodiment, thecosmetically-acceptable topical carrier is from about 50% to abut99.99%, by weight, of the composition (e.g., from about 80% to about95%, by weight, of the composition.

The compositions may be made into a wide variety of product types thatinclude but are not limited to lotions, creams, gels, sticks, sprays,ointments, cleansing liquid washes and solid bars, shampoos, pastes,foams, powders, mousses, shaving creams, wipes, patches, nail lacquers,wound dressing and adhesive bandages, hydrogels, films and make-up suchas foundations, mascaras, and lipsticks. These product types maycomprise several types of cosmetically-acceptable topical carriersincluding, but not limited to solutions, emulsions (e.g., microemulsionsand nanoemulsions), gels, solids and liposomes. The following arenon-limitative examples of such carriers. Other carriers can beformulated by those of ordinary skill in the art.

The topical compositions useful in the present invention can beformulated as solutions. Solutions typically include an aqueous ororganic solvent (e.g., from about 50% to about 99.99% or from about 90%to about 99% of a cosmetically acceptable aqueous or organic solvent).Examples of suitable organic solvents include: propylene glycol,polyethylene glycol (200–600), polypropylene glycol (425–2025),glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol,ethanol, and mixtures thereof.

Topical compositions useful in the subject invention may be formulatedas a solution comprising an emollient. Such compositions preferablycontain from about 2% to about 50% of an emollient(s). As used herein,“emollients” refer to materials used for the prevention or relief ofdryness, as well as for the protection of the skin. A wide variety ofsuitable emollients are known and may be used herein. Sagarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32–43 (1972)and the International Cosmetic Ingredient Dictionary and Handbook, eds.Wenninger and McEwen, pp. 1656–61, 1626, and 1654–55 (The Cosmetic,Toiletry, and Fragrance Assoc., Washington, D.C., 7^(th) Edition, 1997)(hereinafter “ICI Handbook”) contains numerous examples of suitablematerials.

A lotion can be made from such a solution. Lotions typically comprisefrom about 1% to about 20% (e.g., from about 5% to about 10%) of anemollient(s) and from about 50% to about 90% (e.g., from about 60% toabout 80%) of water.

Another type of product that may be formulated from a solution is acream. A cream typically comprises from about 5% to about 50% (e.g.,from about 10% to about 20%) of an emollient(s) and from about 45% toabout 85% (e.g., from about 50% to about 75%) of water.

Yet another type of product that may be formulated from a solution is anointment. An ointment may comprise a simple base of animal or vegetableoils or semi-solid hydrocarbons. An ointment may comprise from about 2%to about 10% of an emollient(s) plus from about 0.1% to about 2% of athickening agent(s). A more complete disclosure of thickening agents orviscosity increasing agents useful herein can be found in Sagarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72–73 (1972)and the ICI Handbook pp. 1693–1697.

The topical compositions useful in the present invention formulated asemulsions. If the carrier is an emulsion, from about 1% to about 10%(e.g., from about 2% to about 5%) of the carrier comprises anemulsifier(s). Emulsifiers may be nonionic, anionic or cationic.Suitable emulsifiers are disclosed in, for example, U.S. Pat. No.3,755,560, U.S. Pat. No. 4,421,769, McCutcheon's Detergents andEmulsifiers, North American Edition, pp. 317–324 (1986), and the ICIHandbook, pp.1673–1686.

Lotions and creams can be formulated as emulsions. Typically suchlotions comprise from 0.5% to about 5% of an emulsifier(s). Such creamswould typically comprise from about 1% to about 20% (e.g., from about 5%to about 10%) of an emollient(s); from about 20% to about 80% (e.g.,from 30% to about 70%) of water; and from about 1% to about 10% (e.g.,from about 2% to about 5%) of an emulsifier(s).

Single emulsion skin care preparations, such as lotions and creams, ofthe oil-in-water type and water-in-oil type are well-known in thecosmetic art and are useful in the subject invention. Multiphaseemulsion compositions, such as the water-in-oil-in-water type, asdisclosed in U.S. Pat. Nos. 4,254,105 and 4,960,764, are also useful inthe subject invention. In general, such single or multiphase emulsionscontain water, emollients, and emulsifiers as essential ingredients.

The topical compositions of this invention can also be formulated as agel (e.g., an aqueous, alcohol, alcohol/water, or oil gel using asuitable gelling agent(s)). Suitable gelling agents for aqueousand/oralcoholic gels include, but are not limited to, natural gums, acrylicacid and acrylate polymers and copolymers, and cellulose derivatives(e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitablegelling agents for oils (such as mineral oil) include, but are notlimited to, hydrogenated butylene/ethylene/styrene copolymer andhydrogenated ethylene/propylene/styrene copolymer. Such gels typicallycomprises between about 0.1% and 5%, by weight, of such gelling agents.

The topical compositions of the present invention can also be formulatedinto a solid formulation (e.g., a wax-based stick, soap bar composition,powder, or a wipe containing powder).

Liposomal formulations are also useful compositions of the subjectinvention. In one embodiment, the peptide and/or the pigment arecontained within the liposome. Examples of liposomes are unilamellar,multilamellar, and paucilamellar liposomes, which may or may not containphospholipids. Such compositions can be prepared by first combininghesperetin with a phospholipid, such as dipalmitoylphosphatidyl choline,cholesterol and water according to the method described in Mezei &Gulasekharam, “Liposomes—A Selective Drug Delivery System for theTopical Route of Administration; Gel Dosage Form”, Journal ofPharmaceutics and Pharmacology, Vol. 34 (1982), pp. 473–474, or amodification thereof. Epidermal lipids of suitable composition forforming liposomes may be substituted for the phospholipid. The liposomepreparation may then incorporated into one of the above carriers (e.g.,a gel or an oil-in-water emulsion) in order to produce the liposomalformulation. Other compositions and uses of topically applied liposomesare described in Mezei, M., “Liposomes as a Skin Drug Delivery System”,Topics in Pharmaceutical Sciences (D. Breimer and P. Speiser, eds.),Elsevier Science Publishers B.V., New York, N.Y., 1985, pp. 345–358, PCTPatent Application No. WO96/31194, Niemiec, et al., 12 Pharm. Res.1184–88 (1995), and U.S. Pat. No. 5,260,065.

In one-embodiment, the liposome is non-ionic. In one example, theliposome contains (a) glycerol dilaurate; (b) compounds having thesteroid backbone found in cholesterol; and (c) fatty acid ethers havingfrom about 12 to about 18 carbon atoms. In a further embodiment, theliposome comprises glycerol dilaurate, cholesterol,polyoxyethylene-10-stearyl ether, and polyoxyethylene-9-lauryl ether. Inone embodiment, these ingredients are in a ratio of about 38:12:33:17.

In one embodiment, the liposomes are present in the topical compositionin an amount, based upon the total volume of the composition, of fromabout 10 mg/ml to about 100 mg/ml such as from about 15 mg/ml to about50 mg/ml. Methods of preparing liposomes are well known in the art, suchas those disclosed in

The topical compositions useful in the subject invention may contain, inaddition to the aforementioned components, a wide variety of additionaloil-soluble materials and/or water-soluble materials conventionally usedin compositions for use on skin, hair, and nails at theirart-established levels.

Additional Cosmetically Active Agents

In one embodiment, the topical composition further comprises anothercosmetically active agent in addition to the peptides and pigments. Whatis meant by a “cosmetically active agent” is a compound (e.g., asynthetic compound or a compound isolated from a natural source) thathas a cosmetic or therapeutic effect on the skin, hair, or nails,including, but not limiting to, lightening agents, darkening agents suchas self-tanning agents, anti-acne agents, shine control agents,anti-microbial agents, anti-inflammatory agents, anti-mycotic agents,anti-parasite agents, external analgesics, sunscreens, photoprotectors,antioxidants, keratolytic agents, detergents/surfactants, moisturizers,nutrients, vitamins, energy enhancers, anti-perspiration agents,astringents, deodorants, hair removers, firming agents, anti-callousagents, and agents for hair, nail, and/or skin conditioning.

In one embodiment, the agent is selected from, but not limited to, thegroup consisting of hydroxy acids, benzoyl peroxide, sulfur resorcinol,ascorbic acid, D-panthenol, hydroquinone, octyl methoxycinnimate,titanium dioxide, octyl salicylate, homosalate, avobenzone,polyphenolics, carotenoids, free radical scavengers, spin traps,retinoids such as retinol and retinyl palmitate, ceramides,polyunsaturated fatty acids, essential fatty acids, enzymes, enzymeinhibitors, minerals, hormones such as estrogens, steroids such ashydrocortisone, 2-dimethylaminoethanol, copper salts such as copperchloride, peptides containing copper such as Cu:Gly-His-Lys, coenzymeQ10, peptides such as those disclosed in PCT Patent ApplicationWO00/15188, lipoic acid, amino acids such a proline and tyrosine,vitamins, lactobionic acid, acetyl-coenzyme A, niacin, riboflavin,thiamin, ribose, electron transporters such as NADH and FADH2, and otherbotanical extracts such as aloe vera and legumes such as soy beans, andderivatives and mixtures thereof. The cosmetically active agent willtypically be present in the composition of the invention in an amount offrom about 0.001% to about 20% by weight of the composition, e.g., about0.01% to about 10% such as about 0.1% to about 5%.

Examples of vitamins include, but are not limited to, vitamin A, vitaminBs such as vitamin B3, vitamin B5, and vitamin B12, vitamin C, vitaminK, and vitamin E and derivatives thereof.

Examples of hydroxy acids include, but are not limited, to glycolicacid, lactic acid, malic acid, salicylic acid, citric acid, and tartaricacid. See, e.g., European Patent Application No. 273,202.

Examples of antioxidants include, but are not limited to, water-solubleantioxidants such as sulfhydryl compounds and their derivatives (e.g.,sodium metabisulfite and N-acetyl-cysteine), lipoic acid anddihydrolipoic acid, resveratrol, lactoferrin, and ascorbic acid andascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbylpolypeptide). Oil-soluble antioxidants suitable for use in thecompositions of this invention include, but are not limited to,butylated hydroxytoluene, retinoids (e.g., retinol and retinylpalmitate), tocopherols (e.g., tocopherol acetate), tocotrienols, andubiquinone. Natural extracts containing antioxidants suitable for use inthe compositions of this invention, include, but not limited to,extracts containing flavonoids and isoflavonoids and their derivatives(e.g., genistein and diadzein), extracts containing resveratrol and thelike. Examples of such natural extracts include grape seed, green tea,pine bark, and propolis. Other examples of antioxidants may be found onpages 1612–13 of the ICI Handbook.

Other Materials

Various other materials may also be present in the compositions usefulin the subject invention. These include humectants, proteins andpolypeptides, preservatives and an alkaline agent. Examples of suchagents are disclosed in the ICI Handbook, pp.1650–1667.

The compositions of the present invention may also comprise chelatingagents (e.g., EDTA) and preservatives (e.g., parabens). Examples ofsuitable preservatives and chelating agents are listed in pp. 1626 and1654–55 of the ICI Handbook. In addition, the topical compositionsuseful herein can contain conventional cosmetic adjuvants, such as dyes,opacifiers (e.g., titanium dioxide), pigments, and fragrances.

Mineral Water

The compositions of the present invention may be prepared using amineral water, for example mineral water that has been naturallymineralized such as Evian® Mineral Water (Evian, France). In oneembodiment, the mineral water has a mineralization of at least about 200mg/L (e.g., from about 300 mg/L to about 1000 mg/L). In one embodiment,the mineral water comprises at least about 10 mg/L of calcium and/or atleast about 5 mg/L of magnesium.

The composition and formulations containing such compositions of thepresent invention may be prepared using methodology that is well knownby an artisan of ordinary skill.

EXAMPLE 1 Induced Pigmentation in Culture

The peptides Peptide 1 and Peptide 3 and various pigments were tested,alone or in combination, with keratinocyte-melanocyte cultures for theireffect on cell pigmentation, using DOPA staining and computerized imageanalysis. The pigments that were tested were the pigmented naturalextracts Bearberry extract from Alban Muller International (Vincennes,France) and Hedychium spicatum extract sold as Kapur Kachali from AmsarPrivate Limited (Indore, India), copper chloride from Sigma (St. Louis,Mo.) and the water-soluble melanin derivatives Melasyn-100™ from San-marlaboratories, Inc. (Elmsford, N.Y.) and MelanZe™ from Zylepsis (Ashford,Kent, United Kingdom). The pigments were tested at concentrationsranging from 0.0001% (W/V) to 1% (W/V) and the peptides were tested at50 μM.

The assay was conducted in the following manner. Human HaCaTkeratinocytes (Boukamp P., et al., J Cell Biol 106, 3, 761–771 (1988))were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing10% fetal bovine serum (FBS), 4.5 mg/ml glucose, 2 mM L-glutamine, 50U/ml penicillin and 50 μg/ml streptomycin (Life Technologies,Gaithersburg, Md.). Cells were maintained at <80% confluency in 5% CO₂(v/v) and were used in experimental procedures up to culture passage 15.Human primary melanocytes (Clonetics, San Diego, Calif. or CascadeBiologics, Portland, Oreg.) were maintained according to manufacturer'sinstructions. To establish keratinocyte-melanocyte co-cultures, 6×10⁴melanocytes were plated in each well of a 24 well plate and maintainedaccording to manufacturer's instructions.

Melanocytes were rinsed three times with melanocyte growth media withoutPMA and keratinocytes (6×10⁴) were plated to establish the co-culturesin this media. Co-cultures were treated for three days with testpeptides and pigments, and assayed for cell viability and pigment levelon the forth day. Cell viability was assayed using alamarBlue™ (AcumedInternational, West Lake, Ohio) following manufacturer's instructions.No change in viability was observed following three daily treatmentswith all tested material. All in vitro experiments were performed intriplicates, and were repeated at least three times.

Following three daily treatments, the co-cultures were briefly fixed(10% buffered formalin from Fisher Scientific, 15 minutes), washed threetimes with Phosphate-buffered saline (PBS, from Life Technologies) andstained with L-3,4-Dihydroxyphenylalanine (DOPA, from Sigma, St. Louis,Mo.) 0.1% in PBS, for 5 hours at 37° C., followed by two PBS washes andformalin (10%, overnight) fixation. DOPA is a substrate for tyrosinase,therefore an increase in staining represents increased tyrosinaseactivity and pigment production. DOPA-stained monolayers were used forimage analysis. All images were obtained and analyzed with Image ProPlus 3.0 software (Media Cybernetics, Silver Spring, Md.). Parametersmeasured were surface area of stained material within melanocyte andkeratinocytes and the total surface area of the cells in the culture,and the relative pigmented area was calculated. A value of 100% wasassigned to untreated controls, and values of treatment groups werenormalized to their relevant controls. In all experiments there was nodifference between PBS-treated cells and untreated controls. Data arepresented with standard deviation (SigmaPlot® 5.0, SPSS Science,Chicago, Ill.).

Table 1 shows the results of representative co-culture experiments,normalized for their relative controls, demonstrating that thecombinations of peptides and pigments of the present invention enhancedpigmentation. This Table demonstrates the specificity of thecompositions of this invention in inducing pigmentation (e.g.,increasing pigmentation by up to 517%). Forskolin (Sigma, St. Louis,Mo.), a known inducer of pigmentation, served as a positive control anda reference point. Forskolin was administered at 1% (W/V).

TABLE 1 Mean % Compositions Conc. area SD increase Control — 0.029 0.009— Peptide 1 50 μM 0.080 0.011 279 Melasyn-100 ™   0.1% (W/V) 0.056 0.013193 Peptide 1 + 50 μM 0.103 0.019 355 Melasyn-100 ™ 0.0001% (W/V)Peptide 1 + 50 μM 0.150 0.036 517 MelanZe ™ 0.0001% (W/V) CuCl₂  0.001%(W/V) 0.064 0.016 220 Peptide 1 + 50 μM 0.108 0.035 372.4 CuCl₂  0.001%(W/V) Peptide 1 + 50 μM 0.050 0.019 172.4 Hedychium extract   0.1% (W/V)Peptide 3 + 50 μM 0.104 0.032 349 Hedychium extract   0.1% (W/V) Peptide3 + 50 μM 0.056 0.015 193.1 Bearberry extract   0.1% (W/V) Forskolin   1% (W/V) 0.067 0.014 231.0

EXAMPLE 2 Induced Pigmentation In Vivo

Compositions of the present invention were tested for their ability toinduce in vivo pigmentation in skin cells in the following experiment.Dark skinned Yucatan microswine (Charles River, Portland, Me.) werehoused in appropriately sized cages in an environmentally controlledroom with a 12-hour light–12-hour dark photoperiod and supplied withfood and water ad libitum. Animal care was based on the “Guide for theCare and Use of Laboratory Animals”, NIH Publication No. 85-23. Twentyμl of test peptides were applied topically, twice a day, five days/week,for eight or nine weeks, on the dorsum of the swine. Treatments ofindividual swine were always arranged in a head to tail order on oneside, and in a tail to head order on the other side of the animal.Biopsies were taken using standard techniques. All swine studiespresented here had no visual irritation, and histological analysesrevealed no markers of irritation or other pathological signs.

Sections from the skin biopsies were stained with Hematoxylin and Eosin(H&E), or with Fontana-Mason (F&M), using standard procedures (Sheenan DC, Hrapckak B B, eds., Theory and Practice of Histo-technology. p. 223,277, The C.V. Mosby Co., St. Louis (1980)). F&M staining identifiessilver nitrate reducing molecules. In skin, this non-specific stainidentifies primarily melanin. At least three sections per biopsy wereprocessed. Each experiment was repeated at least three times.

Swine were treated with either: the known skin darkening agent forskolin(Sigma) or with a Coleus forskoli extract (ATZ Naturals, EnglewoodCliffs, N.J.) which contains forskolin, as a positive control at 1%(W/V); pigments at 1% or 5% (W/V); peptides of this invention at 250 μMor 500 μM; and combinations of such peptides at 250 μM or 500 μM withpigments at 1% (W/V). Pigments tested include pigmented natural extracts(i.e., Bearberry extract and Hedychium spicatum extract) and syntheticcompounds (i.e., the soluble melanin compound Melasyn-100™). These testcompounds were dissolved in ethanol: propylene glycol 70:30 (v/v). In alimited set of experiments, certain peptide and pigment combinationswere also tested using different vehicles, in order to possibly enhancedelivery, namely liposomes and an aqueous gel system, and the ethanol.Liposomes consisted of glyceroldilaurate/cholesterol/polyoxyethylene-10-stearylether/polyoxyethylene-9-lauryl ether at a ratio of 37.5:12.5:33.3:16.7,and were prepared according to Niemiec, et al., 12 Pharm. Res. 1184–88(1995). The gel delivery system consisted of Sepigel 305 (Polyacrylamide& C13-14 isoparaffin & laureth-7 from SEPPIC, Inc. of Fairfield, N.J.).Sepigel 305 was dissolved in the aqueous formulations at 1–3% (W/V).These delivery systems were also tested in the absence of any pigmentsor peptides.

Histological analysis revealed an increase in pigment deposition inswine treated with compositions of this invention. This increase wasgreater than the darkening induced by forskolin or Coleus forskoliextract, which are known skin darkening agents. Occasionally, anindividual swine did not respond to any composition or even forskolin(the positive control). Such “non-responder” swine composed less than25% of the total swine treated.

F&M-stained histological skin sections were evaluated for the change inpigment deposition within the treated site. Criteria for evaluation weretotal increase in pigment deposition, and the presence of cappedepidermal cells above the basal layer. Table 3 represents the averagevalue of all sites of responsive swine treated with each test material.The scale for evaluation is defined in Table 2.

TABLE 2 Score Description −1 Slight lightening 0 No change 1 Minimalincrease in pigment deposition 2 Increased pigment deposition 3 Strongincrease in pigment deposition, some increase in caps 4 Strong increasein pigment deposition, strong increase in caps

TABLE 3 Compositions Score Control 0 Forskolin (positive control) 1Coleus Forskoli (positive 1 control) Ethanol:polypropylene glycol 0Liposomes −1  Gel 0 Peptide 3 2–3 Peptide 1 1–2 Bearberry Extract 1Hedychium Extract 0 Melasyn-100 ™ 0 Peptide 3 and Melasyn-100 ™ 3–4Peptide 1 and Melasyn-100 ™ 4 Peptide 3 and Hedychium 2 Extract Peptide1 and Hedychium 3 Extract Peptide 3 and Bearberry 3 Extract Peptide 1and Bearberry 3 ExtractThis example demonstrates that treatments with compositions of peptidesand pigments of this invention result in increased pigment productionand deposition in vivo.

EXAMPLE 3 Darkening of Human Skin

Human skin from patients undergoing cosmetic surgeries was obtained withwritten consent within 24 hours. Skin was kept in cooled, moistmicroenvironment prior to dermatoming to 0.4 mm thickness. T cell and Bcell deficient mice (Fox Chase C.B-17-SCID, Taconic, N.Y.),six-week-old, were grafted with an oval piece of skin, approximately0.8×2.0 cm in size. Skin was grafted onto a full thickness skin defectof the shaved mouse and fixed by 6-0 silk. Grafts were covered withVaseline impregnated gauze and covered by occlusive dressing. Sutureswere removed after one week. One month later the human grafts weretreated daily, five days a week, for seven weeks, with 15 μl of testmaterial. At seven weeks the human skin was collected for histologicalanalysis.

The human skins were treated with (i) Peptide 1 (500 μM) combined withthe soluble melanin Melasyn-100™ (1% w/v) in liposomes (20 mg/ml, inPBS), (ii) Peptide 1 (500 μM) combined with Bearberry extract (1% w/v)in liposomes (20 mg/ml, in PBS), (iii) Bearberry extract (1% w/v) inliposomes (20 mg/ml, in PBS), or (iv) liposomes alone without peptide orpigments.

By the fifth week, and more noticeably by the sixth week of treatment,the human skins treated with the peptide and pigment combinations ofthis invention were visibly darker than an untreated skin graft from thesame donor. Sections from the skin biopsies were F&M stained asdescribed in Example 2. At least three sections per biopsy wereprocessed. Histological analysis revealed an increase in pigmentdeposition in the grafts treated with peptide 1 combined with eithermelanin or Bearberry extract.

F&M-stained human skin histological sections were evaluated for thechange in pigment deposition within the treated site. Criteria forevaluation were total increase in pigment deposition, and the presenceof capped epidermal cells above the basal layer, as indicated in Example2. The results of a representative experiment are described in Table 4.

TABLE 4 Composition Score Liposome Vescicle 0 Bearberry Extract 0Peptide 1 and Bearberry 2 Extract Peptide 1 and Melasyn-100 ™ 2–3This example demonstrates that compositions of this invention couldvisibly darken human skin, supported by histological data.

It is understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the claims.

1. A composition comprising (i) a peptide having the formulaR₁>Leu-Ile-Gly-Arg-R₃R₂ each R₁ and R_(2,) independently, is H, C₁₋₁₂ alkyl, C₇₋₁₀phenylalkyl, or C(═O)E₁, where E₁ is C₁₋₂₀ alkyl, C₃₋₂₀ alkenyl, C₃₋₂₀alkynyl, phenyl, 3,4-dihydroxyphenylalkyl, naphthyl, or C₇₋₁₀phenylalkyl; provided that when either R₁ or R₂ is C(═O)E₁, the othermust be H; and R₃ is OH, NH₂, C₁₋₁₂ alkoxy, C₇₋₁₀ phenylalkoxy, C₁₁₋₂₀naphthylalkoxy, C₁₋₁₂ alkylamino, C₇₋₁₀ phenylalkylamino, or C₁₁₋₂₀naphthylalkylamino; or a cosmetically acceptable salt thereof; (ii) apigment; and (iii) a cosmetically-acceptable topical carrier.
 2. Acomposition of claim 1, wherein R₁ is H, R₂ is H or C(═O)E₁ where E₁ isC₁₋₂₀ alkyl, and R₃ is OH or NH₂.
 3. A composition of claim 1, whereinsaid peptide is H₂-Leu-Ile-Gly-Arg-NH₂ (SEQ ID NO: 1),H₂-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 2), Palmitoyl-Leu-Ile-Gly-Arg-NH₂ (SEQID NO: 5), Palmitoyl-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 6),Stearatoyl-Leu-Ile-Gly-Arg-NH₂ (SEQ ID NO: 9), orStearatoyl-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 10), or acosmetically-acceptable salt thereof.
 4. A composition of claim 1,wherein said pigment is melanin or a derivative of melanin, wherein saidderivative of melanin is a polymer comprising monomeric units selectedfrom the group consisting of dihydroxyindole-carboxylic acids,3-aminotyrosine, 3,4-dihydroxybenzoic acid, 3-amino,4-hydroxybenzoicacid, aloin, emodin, alizarin, tyrosine, dihydroxyphenylalanine,4,5-dihydroxynaphthalene-2-sulfonic acid, 3-nitrotyrosine,3-dimethylamino phenol and p-aminobenzoic acid.
 5. A composition ofclaim 3, wherein said pigment is melanin or a derivative of melanin,wherein said derivative of melanin is a polymer comprising monomericunits selected from the group consisting of dihydroxyindole-carboxylicacids, 3-aminotyrosine, 3,4-dihydroxybenzoic acid,3-amino,4-hydroxybenzoic acid, aloin, emodin, alizarin, tyrosine,dihydroxyphenylalanine, 4,5-dihydroxynaphthalene-2-sulfonic acid,3-nitrotyrosine, 3-dimethylamino phenol and p-aminobenzoic acid.
 6. Acomposition of claim 1, wherein said pigment is CuCl₂ , Hedychiumextract, or Bearberry extract.
 7. A composition of claim 3, wherein saidpigment is CuCl₂ , Hedychium extract, or Bearberry extract.
 8. A methodof darkening the skin, said method comprising topically applying to theskin a composition of claim
 1. 9. A method of claim 8, wherein R₁ is H,R₂ is H or C(═O)E₁ where E₁ is C₁₋₂₀ alkyl, and R₃ is OH or NH₂.
 10. Amethod of claim 8, wherein said peptide is H₂-Leu-Ile-Gly-Arg-NH₂ (SEQID NO: 1), H₂-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 2),Palmitoyl-Leu-Ile-Gly-Arg-NH₂ (SEQ ID NO: 5),Palmitoyl-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 6),Stearatoyl-Leu-Ile-Gly-Arg-NH₂ (SEQ ID NO: 9), orStearatoyl-Leu-Ile-Gly-Arg-OH (SEQ ID NO: 10), or acosmetically-acceptable salt thereof.
 11. A method of claim 8, whereinsaid pigment is melanin or a derivative of melanin, wherein saidderivative of melanin is a polymer comprising monomeric units selectedfrom the group consisting of dihydroxyindole-carboxylic acids,3-aminotyrosine, 3,4-dihydroxybenzoic acid, 3-amino,4-hydroxybenzoicacid, aloin, emodin, alizarin, tyrosine, dihydroxyphenylalanine,4,5-dihydroxynaphthalene-2-sulfonic acid, 3-nitrotyrosine,3-dimethylamino phenol and p-aminobenzoic acid.
 12. A method of claim10, wherein said pigment is melanin or a derivative of melanin, whereinsaid derivative of melanin is a polymer comprising monomeric unitsselected from the group consisting of dihydroxyindole-carboxylic acids,3-aminotyrosine, 3,4-dihydroxybenzoic acid, 3-amino,4-hydroxybenzoicacid, aloin, emodin, alizarin, tyrosine, dihydroxyphenylalanine,4,5-dihydroxynaphthalene-2-sulfonic acid, 3-nitrotyrosine,3-dimethylamino phenol and p-aminobenzoic acid.
 13. A method of claim 8,wherein said pigment is CuCl₂ , hedychium extract, or bearberry extract.14. A method of claim 8, wherein said pigment is CuCl₂ , hedychiumextract, or bearberry extract.